Intellectual merit: The proposed research explores the fundamental problem of long-term survival of microorganism communities and preservation of biomaterials in fluid inclusions in halite and gypsum. The goal of the proposed research is to determine the distribution, survival, and diversity of microorganism communities and biomaterials that have been in the subsurface for periods of thousands to hundreds of millions of years. The research plan will follow the successful interdisciplinary approach recently used for the study of halite from the subsurface of Death Valley, CA, but extended to older halite deposits, 105 to 108 Ma in age, and for the first time to gypsum. New emphasis will be placed on: (1) Molecular biological techniques involving amplification of fragments of DNA by the polymerase chain reaction (PCR), followed by cloning and sequencing, which will characterize the phylogenetic diversity of microorganisms in fluid inclusions in saline minerals. (2) Raman spectroscopy, which has the potential to characterize the nature of biomolecules in fluid inclusions, in situ. All halite and gypsum to be examined contains preserved primary textures and fluid inclusions originally formed during crystallization in ancient brine bodies. Complementary cultivation experiments will attempt to isolate halophilic and halotolerant Archaea and Bacteria from fluid inclusions in halite and gypsum. Environmental SEM of filtrates from dissolved crystals will help confirm identification of microorganisms and biomaterials made by in situ microscopy and Raman spectroscopy. A second goal of the proposed research will be to attempt to replicate experiments from this project and from published reports claiming amplification of DNA and cultivation of Archaea and Bacteria from ancient halite. Replication of results, an important criterion needed for verifying the authenticity of cultured ancient microorganisms and DNA, will be conducted at a laboratory at the University of Otago, New Zealand, using high throughput next generation DNA sequencing.
Broader Impacts: The proposed research will promote interdisciplinary training and the excitement of scientific discovery at the undergraduate and graduate level in geoscience, anthropology, and biology at Binghamton, Virginia Tech and Otago. Both PIs will incorporate their research into their undergraduate courses, and are committed to K-12 education. Lowenstein?s group will collaborate with the Kopernik Observatory & Science Center ?Link Summer Science Explorations Program? for students in grades 1-12, where research results and hands-on activities related to long-term survival of microbes and preservation of DNA in salt crystals and the implications for life on Mars will be presented. A website will be developed describing the details of the current project, including a searchable database of phylogenetic sequences from organisms sequenced in this project.
The research explored the fundamental problem of long-term survival of microorganism communities and preservation of biomaterials in tiny droplets of water, called fluid inclusions, trapped within crystals of halite and gypsum. We examined the preservation of microorganisms, including prokaryotes and eukaryotes, as well as DNA and other biomolecules. We used: (1) Microscopic techniques to identify microbial remains in fluid inclusions in halite and gypsum, including halophilc Archaea and single celled algae, (2) Molecular biological techniques involving amplification of fragments of DNA by the polymerase chain reaction (PCR), followed by cloning and sequencing, which characterized the phylogenetic diversity of microorganisms in fluid inclusions in saline minerals, and (3) Raman spectroscopy, which characterized the nature of biomolecules in fluid inclusions, in situ. The goal of the research was to obtain data on the distribution, survival, and diversity of microorganism communities and biomaterials that have been buried beneath the Earth’s surface in salt deposits. We recovered 16S ribosomal DNA sequences from subsurface halite, Death Valley, California, 22,000 to 34,000 years old, that are identical, or nearly so, to two strains of halophilic Archaea which were previously cultured from the same halite samples. These results provide the best evidence to date for the long-term survival of halophilic Archaea in ancient halite. This approach can be used to investigate DNA trapped in older halite, millions to hundreds of millions of years in age. Such studies of microbial life in ancient salt are important as we search for microbial signatures in similar deposits on Mars and elsewhere in the solar system. Carotenoids are common in many photosynthetic organisms, and are of great interest as biomarkers in extraterrestrial samples. In this project we identified carotenoids using laser Raman spectroscopy in fluid inclusions from subsurface halite, 9,000 to 1.44 million years in age, from Death Valley, Saline Valley and Searles Lake, California. Carotenoids in fluid inclusions are associated with algal cells similar in appearance to the common halophilic alga Dunaliella. We found that carotenoids are well preserved in ancient salt, which supports other observations that fluid inclusions in buried halite deposits preserve intact halophilic microbial ecosystems. This work demonstrates the value of laser Raman spectroscopy in extraterrestrial exploration for remnants of microbial life. Our research has shown that DNA is preserved in fluid inclusions in crystals of halite for more than 100,000 years. We found in an earlier study that halophilic Archaea can remain alive inside fluid inclusions in halite for at least 34,000 years. Carotenoids, produced by halophilic Archaea and algae, are preserved in halite for more than 1 million years. These findings show that microbial life, some viable, exists in ancient subsurface salt deposits on Earth. Such knowledge is broadening our views about the limits of life on Earth and in other parts of the solar system. Our findings on the long-term survival of prokaryotes and preservation of ancient DNA in crystals of halite will help define target samples in the search for life elsewhere in the solar system. In particular, probable salt deposits on the surface and shallow subsurface of Mars may be fruitful targets when searching for evidence of past life there.
